Epithelial cells form highly polarized sheets that line many internal organs. In adult polycystic kidney disease and in acute ischemic renal injury, changes in cell polarity or frank loss of polarity are observed. A complex of Par3, Par6, atypical PKC (aPKC) is a master regulator of polarization. The Mostov lab has used Madin-Darby canine kidney (MDCK) cells in 3-dimensional collagen gels as a model system to study cell polarity. Under these conditions, the cells form hollow cysts lined by a monolayer of cells, all of which have the apical surface facing the central lumen. The goal of these studies is to understand the role of Par3, Par6, aPKC as well as Cdc42 in the development of polarized cyst structures. In polarized cysts, Par3 is localized to tight junctions, and aPKC is found on the apical surface of cells. To complete the localization studies of these proteins during cyst formation, a GFP-tagged Par6 will be localized by confocal microscopy. aPKC/Par3/Par6 complexes will be further characterized by co-immunoprecipitation studies. Next, changes in the subcellular localization of Cdc42 during cyst formation and the generation of polarity will be examined. Finally, the roles of Par3, Par6 and aPKC in orientation of polarity will be examined. Dominant negative mutants of each of these proteins, as well as control constructs will be expressed in MDCK cells, and the effects of these proteins on polarity in MDCK cells grown in 3 dimensional culture will be examined.